Ultrafast light-induced dynamics in the microsolvated biomolecular indole chromophore with water

Interactions between proteins and their solvent environment can be studied in a bottom-up approach using hydrogen-bonded chromophore-solvent clusters. The ultrafast dynamics following UV-light-induced electronic excitation of the chromophores, potential radiation damage, and their dependence on solvation are important open questions. The microsolvation effect is challenging to study due to the inherent mix of the produced gas-phase aggregates. We use the electrostatic deflector to spatially separate different molecular species in combination with pump-probe velocity-map-imaging experiments. We demonstrate that this powerful experimental approach reveals intimate details of the UV-induced dynamics in the near-UV-absorbing prototypical biomolecular indole-water system. We determine the time-dependent appearance of the different reaction products and disentangle the occurring ultrafast processes. This approach ensures that the reactants are well-known and that detailed characteristics of the specific reaction products are accessible – paving the way for the complete chemical-reactivity experiment.


SUPPLEMENTARY NOTE 1: SPECIES SEPARATION
Using the electrostatic deflector, we spatially separated indole(H 2 O) from the other species present in the molecular beam [1][2][3][4], as shown in Supplementary Figure 1 a. The orange line shows the molecular beam profile with the deflector switched off. In this case, the profiles are practically identical for all molecular species in the beam. However, when the deflector is switched on by applying high voltage to the electrodes, the molecules experience the resulting strong inhomogeneous electric field and are spatially dispersed according to their effective dipole moments. The red and blue lines show the molecular beam profiles for indole and indole(H 2 O), respectively, when the deflector is switched on. These profiles were corrected for the dissociative ionisation of indole(H 2 O) + leading to indole + [3,5]. Here, the lower edge of the indole profile is shifted by ∼1 mm whereas for indole(H 2 O) it is shifted by ∼1.8 mm, reflecting their different dipole moments of 1.96 D and 4.4 D, respectively [6], with respect to their masses. The blue arrow indicates the position used in the dynamics measurements in this work, where we had an indole(H 2 O) sample with an estimated purity of >90%.
Using higher energy laser pulses, we also measured vertical molecular beam profiles for H 2 O (E i = 12.621 eV [7]) and (H 2 O) 2 (E i ≤ 11.21 eV [7]), which have a significantly higher ionisation energy E i than indole(H 2 O) (E i = 7.37 eV [8]) and indole (E i = 7.76 eV [7]). The indole(H 2 O) signal for this higher laser power, corrected for dissociative ionisation (vide supra), is shown in blue in Supplementary Figure 1 b. For improved visibility, the H 2 O + (green) and (H 2 O) + 2 (purple) signals are multiplied by factors of 7 and 22, respectively. For the (H 2 O) 2 signal, we only took low-kinetic-energy ions into account, which result from single ionisation of (H 2 O) 2 . As can be seen from Supplementary Figure 1 b, (H 2 O) 2 deflects even further than indole(H 2 O), due to the higher dipolemoment-to-mass ratio [9].

SUPPLEMENTARY NOTE 3: INSTRUMENT RESPONSE FUNCTION
To determine the temporal overlap of UV and NIR pulses, i. e., t 0 , and the temporal instrument response function (IRF) we performed two calibration experiments. Supplementary Figure 2 a shows the H 2 O + signal as a function of the pump-probe delay in the centre of the molecular beam profile, Y = 0 mm, and the centre of the VMI, KE < 120 meV, which corresponds to H 2 O + signal from single ionisation of H 2 O. Fitting a Gaussian to this cross-correlation of the UV and NIR pulses yields an IRF of τ IRF = 416 fs. Assuming that this H 2 O + signal is due to single ionisation of H 2 O using 1 UV and 9 NIR photons [10] and with an NIR probe-pulse duration of 70 fs (FWHM of the intensity envelope, FWHM I ) from an autocorrelation measurement, a UV pulse duration of 692 fs (FWHM I ) is extracted. The mean of the fitted Gaussian yields t 0 .
These results were confirmed through a recording of the steep increases in the indole(H 2 O) + and indole + signals with high temporal resolution. The IRF can be determined from the incoherent and coherent limits of the fast increases [11]. For the incoherent limit, which in our experiment is the case for indole(H 2 O) with a dephasing time of 20 . . . 100 fs [12], much smaller than the UV pulse duration, the signal is given by I incoherent ∝ 1+erf(t/τ IRF ) [11]. The increase in the indole + signal, however, mostly comes from the πσ * state, which has a large dephasing time. This allows us to approximate this increase by the coherentlimit formulation I coherent ∝ [1 + erf(t/τ IRF )] 2 [11]. A joint fit of I incoherent to the indole(H 2 O) + signal and of I coherent to the indole + signal, Supplementary Figure 2  States 2 (green) and 3 (orange), corresponding to the ππ * and πσ * states of indole(H 2 O), respectively, contribute to the fast increases in the indole + and indole(H 2 O) + signals. The delayed increase of the indole + with respect to the indole(H 2 O) + signal is due to the small contribution of state 2 to the indole signal. When the population in state 2 decreases we observe a fast decrease in the indole(H 2 O) + signal. These observations led us to conclude that the fragmentation probability after ionisation from the πσ * state, state 3, is larger than from the ππ * state, state 2. We ascribe this to a higher-energy cationic state that needs to be reached from the πσ * state, which is most likely energetically above the dissociation energy of the cation, as described in the main manuscript. In that case, four probe photons are needed to reach this cationic state, instead of three for ionisation from the ππ * state. A similar effect was observed for indole(NH 3 ): For a probe wavelength of 395 nm the ππ * state was mostly ionised by a single photon, whereas two photons were needed for ionising the πσ * state [13]. The slow decay of the population in state 3 results in a slow decrease in the indole(H 2 O) + signal. Due to the contribution of state 4, i. e., the S 0 state of indole(H 2 O), however, there is still signal left at the longest delays we measured. We assumed that state 5 has no contribution to the indole(H 2 O) + signal, since this state corresponds to dissociation into separate indole and H 2 O molecules. For indole + the contributions of states 4 and 5 result in an overall slow increase in the signal.

SUPPLEMENTARY NOTE 5: OSCILLATIONS DUE TO WAVEPACKET DYNAMICS
We performed an additional high-temporal-resolution measurement for the indole + signal to investigate the oscillatory structures we observed, as shown in In order to examine the damping of the oscillations, we compared the experiment with the function y(t) = A + B cos(ωt + ϕ). We fitted this function to our data points for t<10 ps and found that A = 0.12, B = 0.03, ϕ = −1.0 and ω = 2π · 0.60 THz, corresponding to 20 cm −1 and T = 1.67 ps. This function is shown by the blue line in Supplementary Figure 4 for short delays. Due to the overall slow increase in the indole + signal for longer delays, A depends on the pump-probe delay. To account for this, we show the same function but with A = 0.14 for the long delays in Supplementary Figure 4. This clearly shows that the oscillations are hardly damped, if at all, on a timescale of 120 ps. We also observed these oscillations with a period of T = 1.67 ps in the indole + signal coming from bare indole (not shown).
However, as these oscillation frequencies are similar for indole, indole(H 2 O), and indole(NH 3 ) and as the oscillations are hardly damped over >100 ps, we assign them to coherent wavepackets of vibrational modes in indole that are not directly involved in the dissociation process, but modulate the ionisation probability for our probe step. In bare indole, there are two in-plane modes with observed vibrational frequencies of 1489 and 1510 cm −1 and several out-of-plane modes, e. g., with observed frequencies of 715, 738, and 762 cm −1 , which all exhibit an energy spacing of ∼20 cm −1 [15] and are, therefore, consistent with our observations. Other possible candidates for these dynamics would be the C-H stretching vibrations with fundamental excitation wavenumbers of 3031, 3054, 3070, and 3091 cm −1 for indole(H 2 O) [16] and at 3051, 3072, and 3090 cm −1 for indole [17] that all exhibit energy spacings of ∼20 cm −1 .
The bandwidth of our UV pulses of 4.5 nm (FWHM), see "Methods", corresponding to 565 cm −1 , allowed to excite wavepackets over many of these states. The effective NIR bandwidth of ∼850 cm −1 is sufficient to probe the complete energy range in a 3-photon ionisation process.